Normal human gingival keratinocyte (NHGK) cells undergo terminal differentiation upon reaching confluency in high Ca++ medium in a similar fashion as in vivo. Induction of TGase 1 activity (5 to 10-fold increase) was followed by formation of insoluble cell envelopes (CEs) suggesting that TGase 1 is a key marker for terminal differentiation of oral keratinocytes. The mRNA levels of other markers of terminal differentiation, e.g. involucrin, SPR1 and annexin 1, were also increased in the terminally differentiating NHGK cells. SPR1, annexin 1, loricrin, envoplakin, desmoplakin, involucrin, cystatin alpha and pancornulin were identified as components of NHGK CEs. These CEs contained an unusually high amount of SPR1, suggesting an important role of SPR1 in the specialized barrier function of oral epithelium. We have compared the expression of cytokeratins 1, 5, 8, 10, 14 and 19, TGase I and TGase II in NHGK cells and several HNSCC (head and neck squamous cell carcinoma) lines including HN4, HN12, HN8, HN22, HN30, HN31, HN13 and HN19. The expression of the major cytokeratins (1, 5, 10 and 14) was significantly reduced in most HNSCC lines. Aberrant expression of cytokeratin 8 or 19 was observed in some carcinoma cells. In contrast to NHGK cells, HNSCC cells failed to induce TGase 1 and failed to form cornified cell envelopes, consistent with their loss of capacity for terminal differentiation. TGase 2 was not inducible but an unusually high level of TGase 2 was observed in HN12, HN30 and HN31 cells. The TGase 2 activity of these HN cells did not correlate with tumorigenicity in nude mice. The immortalized human gingival keratinocytes (IHGK) show a similar pattern of cytokeratin expression, as do NHGK. Like NHGK cells, IHGK cells, immortalized with pBabe vector encoding HPV 16 E6/E7 genes, undergo terminal differentiation upon reaching post-confluency in a high Ca++ medium. In an effort to understand the genetic and molecular mechanisms involved in oral carcinogenesis, we have conducted gene expression profiling in NHGK, IHGK, HSG (human salivary gland line), SGT (human salivary carcinoma line) and 11 HNSCC lines using cDNA microarrays (NCI-OncoChip) containing 6720 cDNA?s. Analysis is underway to correlate the gene expression patterns with the phenotypes of the cells and to identify genetic changes involved in oral carcinogenesis.